Research Director: Dr. Hisato Kondoh
(Professor, Graduate School of Frontier Biosciences, Osaka University)
Research Term: 1998-2003

Through the five-year venture of the Differentiation Signaling Project, attempts were made to initiate new avenues in the field of developmental biology. Three major approaches were taken: large scale screening of mutant Medaka fish defective in embryogenesis; a Wnt family-focused in-depth investigation of signaling systems; and a modernized revisit to the classical problem of tissue regeneration in amphibians. These approaches are believed to have provided new insight into current as well as long-standing problems of animal development.

Research Results

1. Large scale screening of Medaka mutants.

Aiming at the elucidation of regulations operating in early vertebrate development, we undertook the first large-scale and systematic collection of Medaka mutants in this project. Medaka fish is the second vertebrate species after zebrafish employed for a mass screening of developmental mutants. 260,000 mutagenized fish embryos were screened for developmental abnormalities and 1,600 homozygous lethal mutations were identified. Two hundred and fifty mutants were selected for further study, which included a variety of mutants defective in brain development which had not been discovered in zebrafish or mice.

2. Medaka mutants affecting brain regionalization.

Utilizing Medaka mutants, we investigated the mechanisms to establish brain regions in the early embryo which later develop into functional brain parts. Special focus was on identification of the genes defective in the mutants and analysis of the fate determination process among brain-forming cells. Phenotype of the mutants suggests that affected genes include those involved in generating specific characters of a brain region and in producing a boundary between the regions.

3. Cellular basis of brain development in Medaka.

To take advantage of the Medaka embryo and its mutants, a modern analysis of brain development at the cellular level is essential. One approach along this line was to follow many vitally labeled single cells in developing embryos to analyze how cells move and are organized into groups corresponding to specific brain regions. Another was concerned with the neural network after brain regions are established. Fine histological techniques as well as transgenic Medaka lines labeling specific nerves were utilized.

4. New zebrafish mutants.

Zebrafish has its own advantages. Early development of animals fully depends on factors derived from mother, rather than the genes activated only late after fertilization. Identification of mutants of maternal factors involves more complex genetic procedure than ordinary (zygotic) mutants, and few such attempts have been made. We sought to isolate a new class of zebrafish mutants having defects in these maternal factors. Important class of maternal factor mutants belonged to those failing in all cell division after fertilization. We also isolated new zebrafish mutants with affected tail bud and somite development indicative of a defect in signaling system.

5. Wnt signaling protein.

We investigated Wnt proteins, a central player of the signaling process of cell differentiation, at various levels from molecules to tissues. Once synthesized, they undergo unique structural changes before being secreted outside of the cells. Malfunctioning of genes related to Wnt, either Wnt proteins themselves, their receptors or intracellular signaling components, generates signaling problems between cells, and causes abnormal cell differentiation. Some of the somite/tailbud mutants of zebrafish may have such problems. Studies found that the somite of zebrafish depend on the Wnt proteins. A new discovery was made that Wnt signals regulated in various ways the development of the central nervous system, cell growth, regulation of differentiation of the neural stem cells, and determination of the neuron types depending on the context of the events.

6. Potential of differentiation transition

Amphibians, newts and frogs, are rich sources of the phenomenon of differentiation transition. Lens regeneration from completely differentiated iris tissue in newt and from cornea in clawed frog has a research history of over a century, but the key molecular process has remained an enigma. We took advantage of the lens regeneration phenomenon to understand the differentiation transition phenomenon by employing modern approaches. Transgenic newts, for instance, were developed for the first time in the world, and utilized in this research. It was confirmed that the same cell differentiation program operated in lens regeneration as in normal development. An early signaling system to initiate regeneration was also identified.

Fig. 1
Male and female pairs of Medaka (top, the fish on the left is female) and zebrafish (bottom, the fish on the right is female).

Fig. 2
A large scale aquarium system used in mutant screening and mutant analysis.